Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF for such EJ. However, weakening XLF via disrupting interaction interfaces causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

典型的非同源末端连接 (C-NHEJ) 因子可以组装成一个长程 (LR) 复合物，其中 DNA 末端相距较远，包含 DNAPKcs、XLF、XRCC4、LIG4 和 KU 异二聚体和一个短程 (SR) ) 缺乏 DNAPKcs 的复合物，其末端定位用于连接。由于 SR 复合体可以从头形成，因此 LR 复合体（即 DNAPKcs）对染色体 EJ 的作用尚不清楚。我们检查了染色体钝端 DNA 双链断裂 (DSB) 的 EJ，并发现 DNAPKcs 对于此类 EJ 的重要性明显低于 XLF。然而，通过破坏相互作用界面来削弱 XLF 会导致对 DNAPKcs、其激酶活性和钝性 DSB EJ 的 ABCDE 簇自磷酸化位点的显着要求。相比之下，基因组维持的其他方面对 DNAPKcs 激酶抑制敏感，其方式不会因 XLF 丢失而进一步增强（即抑制同源定向修复和结构变异，以及 IR 抗性）。我们建议 DNAPKcs 需要将弱化的 XLF 定位在 LR 复合物中，该复合物可以转变为钝性 DSB EJ 的功能性 SR 复合物，但对于基因组维护的其他方面也具有不同的功能。